Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product

The alkaline α-galactosidase AtAkαGal3 from Arabidopsis exhibits a dual function where it can synthesize stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 and its D383A mutant with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, reveal four complete subsites (–1 to +3) and a new secondary product-binding site, providing the first representative structure of an alkaline α-galactosidase and a model for the raffinose family of oligosaccharide synthases.


Introduction
-Galactosidases (-d-galactoside galactohydrolases; EC 3.2.1.22), which are widely found in microorganisms, plants, animals and humans, catalyze the removal of the nonreducing -d-galactose moiety from galacto-oligosacharides, polysaccharides, galactolipids and glycoproteins (Henrissat et al., 2001). Fabry disease is a rare genetic disease in humans that is due to a deficiency of -galactosidase A, which is needed to catabolize the -d-galactosyl moiety of sphingolipids in the lysosome (Calhoun et al., 1985;Weidemann et al., 2003). The hydrolytic specificity of -galactosidases enables their application in the food industry to remove raffinose and to increase the yield of sucrose in the sugar beet industry (Shibuya et al., 1995), to improve the gelling properties of galactomannans for use as food thickeners and to degrade the raffinose family of oligosaccharides (RFOs) in food and feed materials and in enzymotherapy (Guimarã es et al., 2001).
Based on their glycoside hydrolase (GH) signatures, -galactosidases and RFO synthases belong to GH families 27 and 36, which are characterized by two conserved motifs: KxD and RxxxD (Henrissat & Romeu, 1995;Henrissat, 1991;Carmi et al., 2003;Lee et al., 2004; http://www.cazy.org/). Plant -galactosidases can be further divided into acidic and alkaline forms, according to their optimal pH value for enzymatic activity. Alkaline -galactosidases have optimal activities at neutral-alkaline pH values, whereas acidic -galactosidases have optimal pH values of 3-6.5 (Lee et al., 2004). Another major difference between these two types of -galactosidase is that the total number of amino acids in alkalinegalactosidases is about twice that in acidic -galactosidases. A phylogenic analysis showed that the amino-acid sequences of alkaline -galactosidases share a higher similarity to those of RFO synthases than to those of acidic -galactosidases (Lee et al., 2004). Both alkaline -galactosidases and RFO synthases are unique to plants. Most studies have focused on acidic -galactosidases (Keller & Pharr, 1996;Dey & Pridham, 1972;Herman & Shannon, 1985). Acidic -galactosidases are involved in the mobilization of seed reserves of RFOs and the degradation of cell-wall galactomannan during seed germination (Corchete & Guerra, 1987). It has been suggested that these enzymes also play a role in cell-wall modification during fruit ripening (Kang & Lee, 2001;Nunan et al., 2001).
The first alkaline -galactosidase was uncovered in the Cucurbitaceae family (Gaudreault & Webb, 1986;Gao & Schaffer, 1999). High alkaline -galactosidase activity was mostly found in roots and immature leaves in the early development stage; the activity declined with increasing leaf maturity in Cucurbita pepo (Gaudreault & Webb, 1986). High activity in an immature organ is due to the translocation of RFOs, the major galactosyl-sucrose oligosaccharides, from their source organs to developing organs such as leaves (Gaudreault & Webb, 1986) and fruits (Gao & Schaffer, 1999). Alkaline -galactosidases can be found in more than one form in the Cucurbitaceae family, with varied substrate specificities. Alkaline -galactosidases from Cucurbita pepo, Cucumis sativus and Cucurbita maxima prefer stachyose to raffinose, whereas in Cucumis melo fruit alkaline -galactosidases of forms I and II exhibit a higher activity towards raffinose and stachyose, respectively (Gao & Schaffer, 1999). The maximum activity of maize alkaline -galactosidase was detected in mature dehydrated, germinating and germinated seeds (Zhao et al., 2006); ZmAGA1, with maximum catalysis at pH 7.5, was solely involved in seed germination. Elevated levels of alkaline -galactosidase gene expression were detected when seed germination in maize was interrupted by heat, cold or dehy-dration (Zhao et al., 2006) and when New Zealand spinach was grown under drought conditions (Hara et al., 2008). A rice chloroplast alkaline -galactosidase (OsAkGal) has been suggested to exhibit a function in degrading digalactosyl diacylglycerol, a major glycolipid in the chloroplast thylakoid membrane, during leaf senescence (Lee et al., 2004(Lee et al., , 2009. The Arabidopsis thaliana (Arabidopsis) genome contains genes for three alkaline -galactosidases (At1g55740, At3g57520 and At5g20250), two RFO synthases (At4g01970 and At5g40390) and four acidic -galactosidases (At3g26380, At5g08370, At5g08380 and At3g56310). Here, the proteins encoded by At1g55740, At3g57520 and At5g20250 are referred to as AtAkGal1, AtAkGal2 and AtAkGal3, respectively (Lee et al., 2004). A BLAST search showed that AtAkGal3 is related to probable galactinol-sucrose galactosyltransferases from various species, such as Arabidopsis lyrata subsp. lyrate (XP_020876652), Camelina sativa (XP_010493135), Capsella rubella (XP_006287127), Raphanus sativus (XP_018444981), Brassica oleracea var. oleracea (XP_013612073.1) and Prunus persica (XP_020424207), with 76-94% sequence identity. Moreover, AtAkGal3 shares sequence identities of less than 30% with all acidic -galactosidases from Arabidopsis and other plants.
To date, only structures of acidic -galactosidases have been reported, including rice acidic -galactosidase (PDB entry 1uas; Fujimoto et al., 2003), chicken -N-acetylgalactosaminidase (PDB entry 1ktb; Garman et al., 2002) and human -galactosidase (PDB entry 1r46; Garman & Garboczi, 2004). The structures of rice acidic -galactosidase and the closely related chicken -N-acetylgalactosaminidase revealed a double-displacement catalytic mechanism and the mode of substrate binding (Fujimoto et al., 2003;Garman et al., 2002). However, no structure of any alkaline -galactosidase or RFO synthase was available prior to this work. Here, we report structures of AtAkGal3 and its D383A mutant in complex with various substrates and products, including the natural substrate/product stachyose, revealing four complete substrate-binding subsites (-1 to +3) at the catalytic site for catalysis. The dual-functional AtAkGal3 was also discovered to exhibit stachyose synthase activity in this work; the new structure of AtAkGal3 thus potentially serves as a model for the study of raffinose/stachyose synthases.

Cloning, expression and purification
Materials and chemicals were obtained from Sigma unless specified otherwise. A. thaliana L. Herynh plants (Col-0) were used in this study. AtAkGal3 full-length open frame sequences (2273 bp) were obtained using database searches and sequence analyses using OsAkGal as a query (Lee et al., 2004). The S3 stage of senescent leaves containing about 30-50% chlorophyll compared with full-grown green rosette leaves (100% chlorophyll) was used for total RNA extraction. Chlorophyll determination was carried out as described previously (Lee & Collins, 2001). Total RNA was isolated using the method described previously (Chang et al., 1993), and an Agilent 2100 Bioanalyzer (ThermoFisher, USA) was used to assess its quality and quantity. First-strand cDNA was synthesized from 1 mg of total RNA using the SuperScript III First-Strand Synthesis System, as described by the manufacturer (ThermoFisher, USA). A DNase (Promega, USA) digestion step was conducted prior to reverse transcription in a total volume of 20 ml. AtAkGal3 full-length open frame sequences (2273 bp) containing KpnI and SalI sites were amplified from 1 ml cDNA solution by 30 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s and extension at 72 C for 1 min in 20 ml PCR reaction mixture. The primer pair used was At5gKpnIF2 (GATCTGGGTACCATGACGA TTAAACCGGCGGT) and At5gnostopSalIR (CTGCAGG TCGACTAACTCAACTTGGATCAGA). The AtAkGal3 full-length open frame sequences were cloned into the expression vector pET-30a(+) (Novagen) at KpnI and XhoI sites with a 6ÂHis tag at the N-terminus and were transformed into Escherichia coli BL21(DE3) cells for expression. The bacteria were cultured at 37 C until the OD 600 reached $0.6 prior to induction with isopropyl -d-thiogalactopyranoside (IPTG; final concentration 0.15 mM) in Luria-Bertani (LB) medium at 20 C for 20 h with shaking, and were then harvested by centrifugation at 8000g for 25 min at 4 C. The cell pellet from 1 l culture was suspended in lysis buffer (35 ml) consisting of 50 mM Tris-HCl pH 8.0 and was subjected to cell disruption by ultrasonication using a pulse cycle of 2 s on and 3 s off with a total duration of 20 min at 40% energy on ice. The soluble protein extract was collected by centrifugation at 12 000g for 30 min at 4 C. The target AtAkGal3 was purified using a nickel-immobilized metal ion-affinity chromatography (Ni-IMAC) column with a low imidazole concentration (50 mM), thus avoiding protein contamination at higher concentrations that affects crystallization, in 20 mM Tris-HCl buffer pH 7.8 containing 500 mM NaCl and was further dialyzed against 20 mM Tris-HCl buffer pH 8.2 containing 1 mM dithiothreitol (DTT) and 1 mM galactose overnight. The eluted AtAkGal3 was concentrated and subjected to size-exclusion chromatography on an S200 column previously equilibrated with the abovementioned dialysis buffer. The target protein was then purified using the same buffer condition as the dialysis buffer to improve the purity.
Two AtAkGal3 mutants, D383A and D447A, were generated by a site-directed mutagenesis method (Quik-Change kit, Stratagene) using the pET-30a-AtAkGal3 recombinant vector as a template. The forward primers for the mutants are as follows: D383A, 5 0 -GGACGGTGTGAAAG TGGCTGTGCAGTGTGTATTGG-3 0 ; D447A, 5 0 -TGATTA GAGCATCAGATGCTTTCTATCCACGGGATCC-3 0 . The D383A and D447A mutants were confirmed by sequencing before expression. After Ni-IMAC purification, both mutants were dialyzed against the same dialysis buffer as wild-type AtAkGal3 protein with and without galactose for various substrate and product complex studies. Selenomethioninelabeled AtAkGal3 (SeMet-AtAkGal3) protein was prepared following the method of Van Duyne (Doublié, 2007).
Briefly, a single colony of recombinant AtAkGal3 in E. coli BL21(DE3) cells was inoculated into 5 ml LB culture at 37 C for 12 h and then inoculated into 100 ml M9 medium in a 250 ml flask and cultured overnight. The bacterial cells from this overnight culture were collected and inoculated into 1 l M9 medium; a mixture of amino acids (100 mg lysine, phenylalanine and threonine; 50 mg isoleucine, leucine and valine; 60 mg SeMet) was added when the OD reached 0.6. After bacterial growth for a further 15 min, 0.15 mM IPTG was added to induce protein production. The rice raffinose synthase from O. sativa L. var. Nipponbare (XP_015621501) was overexpressed, purified and characterized using the protocol described previously (Li et al., 2007).

Activity assay using thin-layer chromatography
The purified proteins, wild-type AtAkGal3 and its D383A and D447A mutants, as well as rice raffinose synthase, at equal amounts of 4 Â 10 À12 mol were incubated with the substrate raffinose at a final concentration of 10 mM at 35 C for 1 h. Each protein reaction and standard oligosaccharides (galactose, sucrose, raffinose and stachyose) were spotted (2 ml) onto a silica thin-layer plate (Merck Millipore); the sample was developed with a solvent consisting of a mixture of chloroform, acetic acid and water in a 6:7:1 ratio by volume (Farag, 1978). The results of the reaction were examined by spraying ethanol containing sulfuric acid (10%) onto the plate and baking it on a hotplate until brown spots developed.

Crystallization and collection of X-ray data
The purified SeMet-AtAkGal3, wild-type and mutant AtAkGal3 protein fractions from size-exclusion chromatography were concentrated to 40-50 mg ml À1 in a buffer consisting of 20 mM Tris-HCl pH 8.2, 1 mM DTT with or without 1 mM galactose before crystallization, which was performed using a microbatch method. The protein crystals appeared in 2-3 months using the best crystallization condition (0.1 M ammonium sulfate, 0.3 M sodium formate, 0.1 M Tris pH 7.8, 10% PEG 2000) at 18 C. Among the crystallization trials with various AtAkGal3 proteins, crystals of SeMet-AtAkGal3, wild-type AtAkGal3 and the D383A mutant (referred to in the following as D383A) in various substrate or product complexes could be obtained. No satisfactory crystals of the D447A mutant were obtained. In addition, without galactose neither wild-type nor mutant crystals could be obtained. Crystals of D383A-substrate complexes were obtained by co-crystallization and subsequent crystal soaking with various substrates, including galactinol, galactose, raffinose and stachyose (final concentration 5 mM), in the reservoir solution at 25 C for 10 min.
The crystals were harvested, transferred to reservoir solution containing glycerol (20%) as a cryoprotectant and immediately cooled with liquid nitrogen for data collection. The X-ray diffraction experiments were performed at various wavelengths ranging from 0.9 to 1.0 Å on synchrotron beamlines TPS 05A (with a Rayonix MX300HS detector) and TLS 15A (with a Rayonix MX300HE detector) at the National research papers Synchrotron Radiation Research Center (NSRRC) in Taiwan and on BL44XU (with a Dectris EIGER X 16M detector) at SPring-8 in Japan. The data sets for SeMet-AtAkGal3, AtAkGal3-galactose, D383A-galactose, D383A-stachyose and D383A-galactose-sucrose collected on the TPS 05A and TLS 15A beamlines were processed using HKL-2000(Otwinowski & Minor, 1997 and the D383A-galactinol and D383A-rafffinose data sets collected on the BL44XU beamline were processed with XDS (Kabsch, 2010).

Structure determination and refinement
The initial crystal structure of wild-type AtAkGal3 was determined by the selenomethionine multi-wavelength anomalous diffraction (Se-MAD) phasing method. 29 selenomethionine sites were first located using SOLVE (Terwilliger & Berendzen, 1999) to generate reasonable initial phases with a figure of merit of 0.34 for data in the resolution range 30-3.0 Å . The phases were further calculated and improved with AutoSol (Zhao et al., 2006) in Phenix and the direct phase selection method (Chen et al., 2014). A model of most of the structure was autobuilt with ARP/wARP (Langer et al., 2008), and further model building and adjustment were undertaken with Coot (Emsley et al., 2010). The structures of the D383A mutant in its various complexes were determined by molecular replacement with MOLREP (Vagin & Teplyakov, 2010) from the CCP4 suite  using the previously solved structure of wild-type AtAkGal3 as a model. All refinements were performed with REFMAC  from the CCP4 suite. The structural models of galactose, galactinol, raffinose and starchyose were obtained from eLBOW (Moriarty et al., 2009) in Phenix before building and refinement.
2.4.1. Model validation. The final refined structures of wild-type AtAkGal3 and the various D383A-substrate and D383A-product complexes were validated with MolProbity (Williams et al., 2018). The ligand-binding interaction at the catalytic binding site of the AtAkGal3 structures was analyzed by PLIP (Salentin et al., 2015).

Protein purification
Both the recombinant wild-type AtAkGal3 and the D383A mutant were passed through an Ni-IMAC column and a size-exclusion column (S200) for purification. All purified AtAkGal3 and mutant proteins showed a single band corresponding to a molecular mass of $82 kDa on SDS-PAGE. The target proteins eluted from size-exclusion chromatography at a molecular mass in the range 80-100 kDa, indicating that the biological unit of AtAkGal3 is a monomer.

Hydrolytic activity assay
A hydrolytic reaction mixture containing AtAkGal3 and its natural substrate raffinose was incubated and subsequently analyzed by thin-layer chromatography (TLC; Supplementary  Fig. S1). For a comparison of activity, a similar assay was simultaneously performed with purified recombinant rice raffinose synthase (O. sativa L. var. Nipponbare; Li et al., 2007). After the hydrolysis reactions with rice raffinose synthase and with AtAkGal3, the TLC plate showed that the raffinose substrate was hydrolyzed to galactose and sucrose (lane 5) and to galactose, sucrose and stachyose (lane 6), respectively, while excess raffinose substrate remained. The results indicate that the hydrolase activity of AtAkGal3 is greater than that of rice raffinose synthase towards raffinose as a substrate. Besides hydrolase activity, AtAkGal3 exhibits stachyose synthase activity by transferring a galactose moiety to the raffinose substrate, in which both the galactose donor and acceptor are raffinose. Moreover, the TLC results showed that AtAkGal3 appears to be incapable of synthesizing raffinose as a raffinose synthase according to an activity comparison between AtAkGal3 and rice raffinose synthase with galactinol as the galactose donor and sucrose as the acceptor (data not shown). The activity assay thus shows that alkaline -galactosidase exhibits a dual function of hydrolase and stachyose synthase activities.

Overall structure of Arabidopsis alkaline a-galactosidase
The crystal structure of wild-type AtAkGal3 was first solved ab initio by the selenomethionine multi-wavelength anomalous diffraction (Se-MAD) method at a resolution of 2.8 Å and was subsequently refined to a high resolution of 2.17 Å using the native crystal with the best diffraction quality, which contains two molecules in the asymmetric unit ( Supplementary Fig. S2a). The crystal structures of complexes of the D383A mutant with galactose, galactinol, raffinose and stachyose were subsequently determined using the wild-type AtAkGal3 structure as a search model by the molecularreplacement method ( Table 1). The crystal structure of AtAkGal3 comprises 724 of a total of 749 amino acids that fold into three domains: N-terminal, catalytic and C-terminal ( Supplementary Fig. S2b). The first four residues are absent due to a lack of electron density. The N-terminal domain (residues 5-187) consists of 13 antiparallel -strands connected by variable loops with an -helix at the end. The catalytic domain (residues 206-518) folds into an (/) 8 -barrel structure with eight parallel -strands placed around a central axis and surrounded by eight -helices, which can be seen in -galactosidases of both the GH27 and GH36 families. The C-terminal domain comprises 16 -strands that form two Greek-key motifs comprising residues 537-653 and 654-749, respectively, which are presumably involved in structural stability. Two regions of residues, 103-119 at the N-terminal domain and 254-263 at the catalytic domain, are disordered without electron density, possibly due to loop flexibility on the protein surface ( Supplementary Fig. S2b).

The catalytic residues and binding sites for various natural substrates
A sequence alignment of acidic -galactosidases from rice (Fujimoto et al., 2003), chicken (Garman et al., 2002), human (Garman & Garboczi, 2004), N. benthamiana (Kytidou et al., 2018), B. fragilis, a Gram-negative bacterium (Joint Center for Structure Genomics, unpublished work), S. cerevisiae (Ferná ndez-Leiro et al., 2010) and T. reesei (Golubev et al., 2004) with known structures indicates the presence of two conserved motifs KXD and RxxxD, where X can be Y, F, L or I and x is any variety of amino acid. The aspartic acid residues in the KXD and RxxxD motifs serve as the nucleophile and the catalytic acid (Fujimoto et al., 2003;Garman et al., 2002), respectively ( Supplementary Fig. S3). A sequence comparison of AtAkGal3 with acidic -galactosidases further confirmed that Asp383 and Asp447 are presumably critical residues that serve as the nucleophile and the catalytic acid/base, respectively. In addition, a sequence alignment of AtAkGal3 with alkaline -galactosidases from tomato (AF512549) and melon (AY114164), seed imbibition protein from Arabidopsis (AtAkGal1) and RFO synthases from japonica rice (XP_015621501), cucumber (AAD02832), soybean (XP_006576826), yoshino cherry (PQQ02596) and clementine (XP_006444535) shows these two residues, Asp383 and research papers Table 1 Data-collection and refinement statistics.
To confirm that these two residues, Asp383 and Asp447 in AtAkGal3, play important roles in catalysis, we generated two single mutants D383A and D447A by replacing Asp383 and Asp447 with alanine by site-directed mutagenesis. The TLC results showed that the hydrolase activity of both mutants towards raffinose was abolished (data not shown).
3.6. Interactions of galactose at the binding site of wild-type AtAkaGal3 and the D383A mutant Before the crystallization of wild-type AtAkGal3, galactose was added to the protein solution via dialysis in order to obtain suitable crystals. Our first solved structure of wild-type AtAkGal3 was thus in complex with galactose at a resolution of 2.17 Å . The galactose moiety, with a chair conformation, is located in the À1 subsite of the catalytic binding site (Fig. 1a). The structure of the AtAkGal3-galactose complex shows that the OH group at C1, the anomeric C atom, of the galactose moiety is in a -anomeric form and points towards the bottom side of the catalytic binding site, forming a direct hydrogen bond (distance 3.06 Å ) to the nucleophile Asp383 and to the OD1 group of the acid/base catalytic residue Asp447 through a water molecule, with distances of 2.66 and 2.87 Å , respectively. Moreover, the OD1 group of Asp447 makes a strong hydrogen bond (distance 2.54 Å ) to the OH2 group of the galactose. The other hydroxyl groups of the galactose are also stabilized by numerous hydrogen-bond interactions with various surrounding side chains of the residues at the À1 binding site, such as Arg443, Lys381, Asp243, Asp244 and Trp307. Furthermore, three water molecules help to stabilize the galactose moiety through water-mediated hydrogen-bonding interactions with Trp78, Lys381, Arg443, Asp447 and Asp479.
The structure of the D383A mutant in complex with galactose was also determined at a resolution of 2.05 Å (Fig. 1b). Interestingly, superposition of the two galactose moieties at the catalytic binding sites of the wild-type AtAkGal3-galactose and D383A-galactose complex structures shows that the galactose moiety in the D383A mutant exhibits a chair form with a slight distortion; the O1 hydroxyl group at the C1 carbon is in an -anomeric form (Supplementary Fig. S5). This particular structural form thus places the O1 hydroxyl group of the galactose in a position nearer the OD2 group of Asp447 to form a direct hydrogen-bond interaction with a distance of 2.87 Å . This effect might be due to the loss of an interaction with the carboxyl group of Asp383, which is replaced by alanine with a short side chain. Except for O1 at the anomeric C atom, which exhibits a conformational alteration from a -form to an -form, the positions of the remaining C atoms and hydroxyl groups of galactose are conserved and make unchanged interactions around the catalytic site compared with the wild-type enzyme. In the D383A structure, two additional water molecules interacting with O1 and O6 of galactose help to stabilize the galactose moiety compared with the wild-type AtAkGal3 structure. Active site of the crystal structure of AtAkGal3. (a) The crystal structure of wild-type AtAkGal3. The detailed interactions of galactose (yellow sticks) in a -anomeric form with residues (yellow sticks) at the À1 subsite are shown. OH1 of galactose makes a hydrogen-bond interaction with the side chain of Asp383 and a water molecule (cyan sphere), with distances of 3.06 and 2.66 Å , respectively. Three water molecules (cyan spheres) help to stabilize the galactose moiety through water-mediated hydrogen-bonding interactions with Trp78, Lys381, Arg443, Asp447 and Asp479. (b) A galactose bound at the catalytic site of the D383A mutant forms an interaction network with residues similar to that in wild-type AtAkGal3, except that there is a direct interaction between OD2 of Asp447 and O1 of the galactose moiety; two additional water molecules (cyan spheres) help to stabilize O1 and O6 of galactose as Asp383 is replaced by alanine.
Using the Cremer-Pople calculator, the galactose molecules complexed in the D383A and D383D mutants show Cremer-Pople parameters ', , Q of 88.829 , 24.021 , 0.713 and 123.014 , 6.867 , 0.646, respectively, which might indicate an induced fit of the substrate to push it towards the transition state.
The residues involved in product-binding interactions with the galactose moiety at the À1 subsite of AtAkGal3 are similar to those in rice acidic -galactosidase (Fujimoto et al., 2003), except for Trp78 in AtAkGal3, which is structurally related to Trp164 of rice acidic -galactosidase and to Trp65 of T. maritima -galactosidase (Ren et al., 2018), which positions its side chain farther away from the galactose moiety. Trp164 of rice acidic -galactosidase, which is located on the loop at the end of the 5 strand, directly interacts with the O1 hydroxyl group of the galactose at the À1 subsite and is a conserved residue among acidic -galactosidases from plants, bacteria and yeast, suggesting that it plays a role in recognizing the galactose substrate moiety (Fujimoto et al., 2003). In contrast, Trp78 of AtAkGal3, which is located in a shortturn (residues 75-78) between two -strands of the N-terminal domain, contributes part of the catalytic binding site together with the catalytic domain. Except for water-mediated interactions with the O1 hydroxyl group of the galactose and the side chain of Asp447, Trp78 seems to make no direct interactions with the galactose moiety at the À1 subsite in AtAkGal3, but rather to make a major interaction at the +1 subsite, which is discussed in the following section.

Interactions of galactinol in the binding site of the D383A mutant
The structure of D383A in complex with galactinol was determined at a resolution of 2.63 Å from D383A crystals by a combination of co-crystallization and soaking with galactinol as described in Section 2. The 5R-OH group (O6 0 ) of the myoinositol moiety of galactinol at the +1 subsite is stabilized by the NE1 group of Trp78 together with OD1 of Asp446 by hydrogen bonds. In addition, 4S-OH is stabilized by the NZ group of Lys75 by hydrogen bonds (Fig. 2a). The conformation of the galactosyl moiety of galactinol differs from that of galactose bound to wild-type AtAkGal3 and the D383A mutant (Fig. 2b), but the galactosyl moiety remains stabilized at the À1 subsite by mostly the same residues, including Asp447, Arg443, Lys381 and Asp244, but not Asp243 and Trp307, making interactions with galactose. Two water molecules are located in the catalytic binding site, one of which interacts with the galactosyl moiety and the other with the myo-inositol moiety and the side chain of Asp346, to help stabilize the galactinol. This Asp346 residue seems to play a major role in galactinol binding at the +1 subsite (Fig. 2a).

Interactions of raffinose in the binding site of the D383A mutant
The crystal structure of D383A in complex with raffinose was determined at a resolution of 2.47 Å . Lys75, Tyr78, Asp446 and Tyr449 help to stabilize the glucose moiety of the raffinose substrate at the +1 subsite (Fig. 3). The NE1 group of Trp78 and OD1 of Asp446 form hydrogen bonds to O4 of the hydroxyl group with distances of 3.08 and 3.47 Å , respectively.  Interactions of galactinol at the active site of the D383A mutant compared with galactose binding to Asp383 in the wild type. The structure of D383A in complex with galactinol is shown. (a) The galactosyl moiety of galactinol (yellow sticks) is bound at the À1 subsite. OD2 of Asp447 makes a direct interaction with O2 of the galactosyl moiety at a distance of 3.36 Å . Lys75, Trp78, Asp346, Asp446 and Tyr449 (green sticks) are involved in stabilization of the myo-inositol moiety by hydrogen-bonding interactions at the +1 subsite. Water molecules are shown as cyan spheres. (b) The carbon ring of the galactosyl moiety (yellow) of galactinol is distorted from that of galactose (green) bound to Asp383; the O3 and O4 groups hence interact with a different residue, Lys381, at the catalytic site as shown in (a). NZ of the Lys75 side chain stabilizes O3 of the hydroxyl group through a hydrogen bond with a distance of 3.54 Å . Tyr449 stabilizes the glucose at the +1 subsite and the fructose moiety at the +2 subsite of raffinose via hydrogen bonds from its sidechain OH group to the O3 hydroxyl groups of both galactose and fructose with distances of 3.52 Å (Fig. 3). One water molecule that forms a hydrogen bond (distance of 2.70 Å ) to the side chain of Asp447 seems to be involved in stabilization of the fructose moiety through weak hydrogen bonding. The interactions of the residues at the À1 subsite with the galactosyl moiety of raffinose are similar to those with the galactosyl moiety of galactinol bound in the D383A complex.

Interactions of the galactose and sucrose products of hydrolyzed raffinose in the binding site
During the crystallization of the D383A-raffinose complex, co-crystallization of the D383A mutant and the raffinose substrate was occasionally set up for over two months. Unexpectedly, we observed both the galactose and sucrose products in the catalytic binding site and an additional cavity in the crystal structure after a sufficient reaction time (Fig. 4a). The D383A mutant seems to be capable of gradually hydrolyzing the raffinose substrate into galactose and sucrose products that remain in the catalytic site. The D383A mutant in complex with hydrolyzed raffinose reveals a secondary product-binding site for sucrose; this site is stabilized and formed in part by a unique short loop (residues 329-352) composed of three short helices at the surface of the catalytic pocket, which equip Thr342 and Asp346 to interact with the glucose and fructose moieties of sucrose through hydrogen bonds (Fig. 4b). This distinct extra loop (residues 329-352) in AtAkGal3 can also be found in raffinose/stachyose synthases based on sequence alignment (Fig. 4c), but not in acid -galactosidase-related proteins. Lys75 from a -turn (residues 75-78) in the N-terminal domain also helps to stabilize the glucose and fructose moieties of sucrose by hydrogen bonds with a distance of 2.80 Å . In addition, Trp78 and Asp447 help to stabilize both the sucrose product and the galactose moiety at the À1 subsite through a water molecule (Fig. 4b).
3.10. Interactions of stachyose in the binding site of the D383A mutant The crystal structure of the complex of D383A with stachyose determined at a resolution of 2.0 Å further reveals the complete subsites (-1, +1, +2 and +3) of AtAkGal3 (Fig. 5). The galactose at the +1 subsite of stachyose is stabilized by hydrogen bonds to both NE of the Lys75 side chain and NH1 of the Trp78 side chain with distances of $3 Å . Even though Trp77 makes no direct interaction with galactose at the +1 subsite, it helps to stabilize galactose through a water molecule. The extensive water network through the side chain of Arg451 helps to stabilize the glucose moiety at the +2 subsite through a water molecule. In addition, water-mediated hydrogen-bond interactions greatly stabilize both the glucose and fructose moieties at the +2 and +3 subsites.

Structural and sequence comparison explain the dual function of AtAkaGal3
The GH36 family contains enzymes, including RFO synthases and alkaline -galactosidases, that generally exhibit transglycosylation and hydrolase activities. The first reported -galactosidase structure was that of T. maritima -galactosidase (Ren et al., 2018). Structural alignment of T. maritima -galactosidase with the related GH27 family enzymes shows that T. maritima -galactosidase and rice acidic -galactosidase (Fujimoto et al., 2003) share the greatest structural similarity. A structural comparison of the (/) 8 -barrel domain of our alkaline -galactosidase AtAkGal3 (residues 201-531) with those of rice acidic -galactosidase (residues 6-271) and T. maritima -galactosidase (residues 165-481) shows poor structural similarity, with r.m.s.d.s of 3.74 and 11.37 Å , respectively. The structure of wild-type AtAkGal3 shows that the catalytic residues Asp383 and Asp447 are separated by approximately 6.7 Å , which is also generally found for the double-replacement retaining mechanism in members of clan D of the GH27 enzymes, with an average distance of 6.5 Å , and in T. maritima -galactosidase, with a distance of 6.3 Å (Comfort et al., 2007). A structural alignment of the (/) 8 -barrel domains of AtAkGal3, rice acidic -galactosidase and T. maritima -galactosidase based on the AtAkGal3 structure shows that most catalytic binding residues at the À1 subsite are conserved. In addition, Trp78 of AtAkGal3 is not part of the (/) 8 -barrel of the catalytic domain like the corresponding Trp of rice acidicgalactosidase, but is located in the N-terminal domain like Trp65 of T. maritima -galactosidase. Moreover, Lys75 and The active-site structure of the complex of D383A with raffinose (yellow sticks). Various residues (green sticks) are involved in binding interactions with glucose at the +1 subsite and fructose at the +2 subsite, as well as the galactosyl moiety at the À1 subsite. The catalytic residue Asp447 and the D383A mutation are also shown. One water molecule (cyan sphere) is located at the catalytic binding site to help stabilize the fructose moiety through hydrogen bonding.
Trp78 in the N-terminal domain share hydrogen-bond interactions that stabilize the second galactose moiety in the +1 subsite (Table 2).
A sequence alignment (ESPript3.0; Robert & Gouet, 2014) of plant alkaline -galactosidases with plant raffinose and stachyose synthases shows that the FRSK 75 xW 77 W 78 region (referring to AtAkGal3) located at the -turn between two -strands of the N-terminal domain is conserved among these families (Fig. 6). Furthermore, two residues, Arg451 and Tyr449 of AtAkGal3, which are also found in raffinose and stachyose synthases, help to stabilize the binding of the galactose moiety at the +2 subsite through water molecules and directed interaction, respectively. These observations from both structural and sequence alignments lead to the possibility of a dual function of the alkaline -galactosidase AtAkGal3, as the identified residues involved in product and substrate binding at the À1, +1 and +2 subsites of the alkaline -galactosidase AtAkGal3 are identical to the functional residues in both the GH27 and GH36 families as well as the raffinose and stachyose synthase families. However, the structure of D383A complexed with stachyose shows that water molecules seem to be important to help to stabilize the fructose moiety at the +3 subsite (Table 2).

N-terminal domain and catalytic site of AtAkaGal3
Our work confirms that the N-terminal domain of the alkaline -galactosidase AtAkGal3 is important for protein function because it contributes part of the core (/) 8 -barrel catalytic site for substrate binding. The C-terminal domain helps to stabilize this structural architecture through interactions with both the N-terminal domain and the (/) 8 -barrel  The electron density of galactose and sucrose products at the active site and sequence alignment of the loop region. (a) Both the galactose and sucrose products (yellow sticks) are clearly revealed with electron densities (gray mesh) at the À1 subsite and the secondary product-binding site, respectively, after co-crystallization of D383A and raffinose for two months. The secondary product-binding site is near the surface of the catalytic binding cavity, which is formed in part by a unique loop with three short helices (magenta) and a -turn (residues 75-78). (b) The interaction network with distances between galactose, sucrose (yellow sticks) and residues (green and magenta sticks) is shown. Water molecules are presented as cyan spheres. (c) A sequence alignment shows that the loop region (residues 329-352 in the green box) exists exclusively in AtAkGal3 and possibly in the RFO synthase family from different plants, including rice (ABF99470; Rice_seed_imbibition), soybean (XP_006576826; Raf_Soybean), pea (RFS_PEA; Raf_Pea), jute (OMO49898; Raf_Corchorus), cucumber (AAD02832; Raf_Cucumis), cacao (EOY02480; Raf_Cacao), japonica rice (XP_015621501; Raf_Oryza sativa), melon (NP_001284472; Raf_II_Melon), yoshino cherry (PQQ02596; STA_Prunus), clementine (XP_006444535; STA_Citrus) and Arabidopsis (NP_192106; STA_A. thaliana). Residue numbers are based on AtAkGal3.
domain. Sequence alignment indicates that the RFO synthases might also use their N-terminal domain for a similar function. The stachyose bound in the catalytic pocket of AtAkGal3 shows that the depth and dimensions of the binding pocket can accommodate four sugar moieties, whereas the catalytic binding sites of the acidic -galactosidases rice -galactosidase and T. maritima -galactosidase are relatively shallow and are likely to accommodate only one galactose. Moreover, the extra loop (residues 329-352) that only exists in the alkaline -galactosidase AtAkGal3 provides two key residues Asp346 and Thr342 to help stabilize the fructose and glucose moieties of the sucrose product in the +2 and +3 subsites, respectively (Figs. 4 and 7).
Our D383A mutant can gradually hydrolyze raffinose into galactose and sucrose. A structural superimposition of D383A in complex with raffinose, with stachyose and with hydrolyzed raffinose in the catalytic binding pocket shows that the wider The active-site structure of D383A in complex with stachyose. The complexed structure reveals the complete subsites (-1, +1, +2 and +3) in the catalytic binding site of AtAkGal3. The glucose in the +2 subsite, stabilized by Arg451, Tyr215 and Trp211 through water-mediated hydrogen-bond interactions, is located nearly at the edge of the pocket cavity. The binding interaction of fructose in the +3 subsite, which is located at the outer edge of the cavity, is utilized only by Asp346 (magenta sticks) and one water molecule at the outer edge of the cavity. Stachyose, protein residues and water molecules are shown as yellow sticks, green sticks and cyan spheres, respectively.
Bold labels indicate the key catalytic residues at the active site. Substrates and products: Gal, galactose; Gol, galactinol; Raf, raffinose; Sta, stachyose. Trp164 Asp185 Asp387 Asp binding-site pocket of AtAkGal3 provides a secondary product-binding site to stabilize the sucrose after raffinose has been hydrolyzed into galactose and glucose. This productbinding site contains Thr342 and Asp346 on a short loop (residues 329-352) that interact with the glucose and fructose moieties of sucrose through hydrogen bonds. Lys75 in the N-terminal domain also helps to stabilize the glucose moiety of sucrose by hydrogen bonding (Fig. 8).

Dual function of AtAkaGal3
We compared the hydrolase activities of the alkaline -galactosidase AtAkGal3 and rice (O. sativa L. var. Nipponbare) raffinose synthase towards only the natural substrate raffinose, without galactinol, which serves as a galactose donor for raffinose or stachyose synthase activity. The TLC assay showed that AtAkGal3 exhibits a greater hydrolase activity towards the raffinose substrate considering the TLC spot intensities of the galactose and sucrose products. AtAkGal3 can also produce a stachyose product without galactinol as a galactose donor. This observation indicates that AtAkGal3 seems to act with the transferase mechanism observed for fructosyl transferases and xyloglucan endotransferases. Our crystal structures of wild-type AtAkGal3 and its D383A mutant in complex with galactose and with hydrolyzed raffinose, i.e. galactose and sucrose, reveal one water molecule at the conserved position near the anomeric C atom and Asp447. This water is replaced by O4 of galactose in Superimposition of catalytic (/) 8 -barrel domains. (a) Superimposition of the catalytic (/) 8 -barrel domains of the alkaline -galactosidase AtAkGal3 (green), rice -galactosidase (gray) and T. maritima -galactosidase (yellow) with the product galactose at the respective À1 subsite. An extra loop (residues 329-352, magenta) exists exclusively in AtAkGal3. (b) The complex of D383A with stachyose reveals that an extra loop helps to stabilize the product or stachyose substrate at the +2 and +3 subsites in the alkaline -galactosidase AtAkGal3. An electrostatic surface is shown (blue, positive charge; red, negative charge). (c, d) Docking of stachyose from the D383A complex into the structures of rice -galactosidase (c) and T. maritima -galactosidase (d) shows that both catalytic binding sites are shallow such that they can accommodate only one galactose, compared with AtAkGal3 that can hold four-sugar moieties such as stachyose. the +1 subsite of raffinose and stachyose in the complexes of D383A with raffinose and stachyose. The only difference between the hydrolase and transferase reactions is the acceptor type after the removal of the terminal glycosyl moiety. The acceptor for a hydrolase is a water molecule, whereas a transferase uses a saccharide or an oligosaccharide, such as sucrose, raffinose or stachyose, as the acceptor (Henrissat et al., 2001;Carmi et al., 2003;Chuankhayan et al., 2010). In AtAkGal3, the conserved water molecule identified above might thus serve as an acceptor in the hydrolase step, releasing galactose as a product, while sucrose and raffinose act as an acceptor for the transferase mechanism, releasing raffinose and stachyose as products. In addition, the unique extra loop (residues 329-352) in AtAkGal3, which contributes to the depth enhancement of the catalytic pocket, presumably supports the transferase mechanism to help to stabilize the oligomer products.

Catalytic and synthetic mechanism
The structure of D383A in complex with hydrolyzed raffinose reveals the products galactose at the À1 subsite and sucrose at the secondary binding site. Unexpectedly, after raffinose has been hydrolyzed between galactose and glucose at subsites À1 and +1, respectively, the sucrose shifts and rotates by 180 . The glucose moiety of sucrose in the +1 subsite is thus replaced by a fructose moiety and is bound in a different orientation and position to the related moieties of raffinose and stachyose in other D383A-substrate complexes (Fig. 8). We specify this newly discovered binding position as the secondary product-binding site. A 180 rotation of the sucrose product after the hydrolysis of raffinose is possible because the catalytic pocket is wide, which provides sufficient space for flipping and binding (Fig. 9). After rotation, Thr342 and Asp346 help to stabilize the glucose and fructose of the sucrose product, respectively. Furthermore, Lys75, which helps to stabilize the sugar moiety in the +1 subsite, also makes a strong interaction with the glucose of the sucrose product in the +3 subsite, indicating that the conserved residues Lys75 and Trp78 in the WW box region are important for stabilization of both the substrate and the product in the +1 subsite, according to the proposed mechanism in Fig. 10. Lys381 might be the key stabilizer during the catalytic process.
In summary, the alkaline -galactosidase AtAkGal3 from A. thaliana exhibits a dual function: it not only catalyzes the hydrolysis of -d-galactose from galacto-oligosaccharides under alkaline conditions but can also synthesize stachyose  The active site for galactose and the secondary active site for sucrose. (a) A side view of the superimposed substrate raffinose (magenta) and products galactose and sucrose (cyan) from hydrolyzed raffinose in the catalytic binding pocket shown as an electrostatic surface. An empty space is found when the substrate raffinose is bound; this space is then occupied by the sucrose product after raffinose has been hydrolyzed. (b) A top view clearly shows that after raffinose has been hydrolyzed the sucrose product is flipped 180 and occupies the secondary productbinding site.

Figure 8
Active-site structures of D383A in complex with raffinose. The structures of D383A in complexes with raffinose (magenta), with stachyose (green) and with hydrolyzed raffinose, i.e. galactose and sucrose (cyan), in the catalytic binding pocket shown with the electrostatic surface indicate that the wider binding-site pocket of the alkaline -galactosidase AtAkGal3 provides a new secondary product-binding site to stabilize the sucrose moiety (cyan). using raffinose, instead of galactinol, as the galactose donor. Structural analyses of AtAkGal3 and its D383A mutant in complex with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, elucidate four complete subsites (-1 to +3) and a new secondary product-binding site. The AtAkGal3 structure in this study is the first representative structure of an alkaline -galactosidase and may also serve a structural model for the raffinose family of RFO synthases.

Figure 10
The proposed catalytic mechanism of the alkaline -galactosidase AtAkGal3. Schematic presentation of the proposed catalytic mechanism of the alkaline -galactosidase AtAkGal3 with raffinose as a donor and an acceptor. The main interactions of residues involved in substrate recognition and the catalytic process are shown: the nucleophile (Asp383), acid/base catalyst (Asp447), Lys381 and Arg443 for the À1 subsite, Trp78, Lys75, Asp446 and Tyr449 for the +1 subsite, Lys75, Trp77, Thr342 and Asp346 for sucrose at the secondary product-binding site, Asp346 for the +3 subsite and water molecules that help to stabilize the +2 subsite. (a) Raffinose as a donor substrate binds in the catalytic binding site. (b) After the glycosidic bond of raffinose has been cleaved, the sucrose product rotates 180 and binds to the secondary product-binding site. The sucrose is subsequently released and galactose remains in the catalytic pocket. (c) Another raffinose, which acts as an acceptor substrate, enters the pocket and moves towards the galactose that remains at the À1 subsite. (d) The acceptor raffinose is engaged with galactose, resulting in the production of stachyose.